The present invention relates to nucleotide sequences which encode polypeptides involved in the DNA mismatch repair systems of plants, and to the polypeplides encoded by those nucleotide sequences. The invention also relates to nucleotide sequences and polypeptide sequences for use in altering the DNA mismatch repair system in plants. The invention also relates to a process for altering the DNA mismatch repair system of a plant cell, to a process for increasing genetic variations in plants and to processes for obtaining plants having a desired characteristic.
Plant breeding essentially relies on and makes use of genetic variation which occurs naturally within and between members of a family, a genus, a species or a subspecies. Another source of genetic variation is the introduction of genes from other organisms which may or may not be related to the host plant.
Allelic loci or non-allelic genes which constitute or contribute to desired quantitative (e.g. growth performance, yield, etc.) or qualitative (e.g. deposition, content and composition of seed storage products; pathogen resistance genes: etc.) traits that are absent, incomplete or inefficient in a species or subspecies of interest are typically introduced by the plant breeder from other species or subspecies, or de novo. This introduction is often done by crossing, provided that the species to be crossed are sexually compatible. Other means of introducing genomes, individual chromosomes or genes into plant cells or plants are well known in the art. They include cell fusion, chemically aided transfection (Schocher et al., 1986. Biotechnology 4: 1093) and ballistic (McCabe et al., 1988, Biotechnology 6: 923), microinjection (Neuhaus et al., 1987, TAG 75: 30), electroporation of protoplasts (Chupeau et al., 1989, Biotechnology 7: 53) or microbial transformation methods such as Agrobacterium mediated transformation (Horsch et al., 1985, Science 227: 1229; Hiei et al., 1996, Biotechnology 14: 745).
However, when a foreign genome, chromosome or gene is introduced into a plant, it will often segregate in subsequent generations from the genome of the recipient plant or plant cell during mitotic and meiotic cell divisions and, in consequence, become lost from the host plant or plant cell into which it had been introduced. Occasionally, however, the introduced genome, chromosome or gene physically combines entirely or in part with the genome, chromosome or gene of the host plant or plant cell in a process which is called recombination.
Recombination involves the exchange of covalent linkages between DNA molecules in regions of identical or similar sequence. It is referred to here as homologous recombination if donor and recipient DNA are identical or nearly identical (at least 99% base sequence identity), and as homeologous recombination if donor and recipient DNA are not identical but are similar (less than 99% base sequence identity).
The ability of two genomes, chromosomes or genes to recombine is known to depend largely on the evolutionary relation between them and thus on the degree of sequence similarity between the two DNA molecules. Whereas homologous recombination is frequently observed during mitosis and meiosis, homeologous recombination is rarely or never seen.
From a breeder""s perspective, the limits within which homologous recombination occurs, therefore, define a genetic barrier between species, varieties or lines, in contrast to homologous recombination which can break this barrier. Homeologous recombination is thus of great importance for plant breeding. Accordingly there is a need for a process for enhancing the frequency of homeologous recombination in plants. In particular, there is a need for a process of increasing homeologous recombination to significantly shorten the length of breeding programs by reducing the number of crosses required to obtain an otherwise rare recombination event.
At least in Escherichia coli, homologous and homeologous recombination are known to share a common pathway that requires among others the proteins RecA, RecB, RecC. RecD and makes use of the SOS induced RuvA and RuvB, respectively. It has been suggested that mating induced recombination follows the Double-Strand Break Repair model (Szostak et al., 1983, Cell 33, 25-35), which is widely used to describe genetic recombination in eukaryotes. Following the alignment of homologous or homeologous DNA double helices the RecA protein mediates an exchange of a single DNA strand from the donor helix to the aligned recipient DNA helix. The incoming strand screens the recipient helix for sequence complementary, seeking to form a heteroduplex by hydrogen bonding the complementary strand. The displaced homologous or homeologous strand of the recipient helix is guided into the donor helix where it base pairs with its counterpart strand to form a second heteroduplex. The resulting branch point then migrates along the aligned chromosomes thereby elongating and thus stabilising the initial heteroduplexes. Single stranded gaps (if present) are closed by DNA synthesis. The strand cross overs (Holliday junction) are eventually resolved enzymatically to yield the recombination products.
Although in wild type E. coli homologous and homeologous recombination are thus mechanistically similar if not identical, homologous recombination in conjugational crosses E. colixc3x97E. coli occurs five orders of magnitude more frequently than homeologous recombination in conjugational crosses E. colixc3x97S. typhimurium (Matic et al. 1995; Cell 80, 507-515). The imbalance in favour of homologous recombination was shown to be caused largely by the bacterial MisMatch Repair (MMR) system since its inactivation increased the frequency of homeologous recombination in E. coli up to 1000 fold (Rayssiguier et al. 1989, Nature 342. 396-401).
In E. coli, the MMR system (reviewed by Modrich 1991, Annual Rev Genetics 25, 229-253) is composed of only three proteins known as MutS, MutL and MutH. MutS recognizes and binds to base pair mismatches. MutL then forms a stable complex with mismatch bound MutS. This protein complex now activates the MutH intrinsic single stranded endonuclease which nicks the strand containing the misplaced base and thereby prepares the template for DNA repair enzymes.
During recombination. MMR components inhibit homeologous recombination. In vitro experiments demonstrated that MutS in complex with MutL binds to mismatches at the recombination branch point and physically blocks RecA mediated strand exchange and heteroduplex formation (Worth et al., 1994; PNAS 91, 3238-3241). Interestingly, the SOS dependent RuvAB mediated branch migration is insensitive to MutS/MutL, explaining the observed slight increase in SOS dependent homeologous recombination. Homeologous mating even induces the SOS response, thereby taking advantage of RuvAB induction (Matic et al. 1995, Cell 80, 507-515).
The MMR system thus appears to be a genetic guardian over genome stability in E. coli. In this role it essentially determines the extent to which genetic isolation, that is, speciation, occurs. The diminished sensitivity of the SOS system to MMR, however, allows (within limits) for rapid genomic changes at times of stress, providing the means for fast adaptation to altered environmental conditions and thus contributing to intraspecies genetic variation and species evolution.
The important role of MMR in preserving genomic integrity has been established also in certain eukaryotes. In its efficiency, the human MMR, for example, may even counteract potential gene therapy tools such as triple-helix forming oligonucleotides including RNA-DNA hybrid molecules (Havre et al., 1993, J. Virology 67: 7234-7331; Wang et al., 1995, Mol. Cell. Biol. 15: 1759-1768; Kotani et al., 1996, Mol. Gen. Genetics 250: 626-634; Cole-Strauss et al., 1996, Science 273: 1387-1389). Such oligonucleotides are designed to introduce single base changes into selected DNA target sequences in order to inactivate for example cancer genes or to restore their normal function. The resulting base mismatches however are recognised by the mismatch repair system which then directs removal of the mismatched base, thereby reducing the efficiency of oligonucleotide induced site-specific mutagenesis.
To date, two families of related genes, homologous to the bacterial MutS and MutL genes have been identified or isolated in yeast and mammals (recent reviews by Arnheim and Shibata, 1997, Curr. Opinion Genet. Dev. 7, 364-370; Modrich and Lahue, 1996, Annual Rev. Biochem. 65, 101-133: Umar and Kunkel, 1996, Eur. J. Biochem. 238, 297-307). Biochemical and genetic analysis indicated that eukaryotic MutS homologs (MSH) and MutL homologs (MLH, PMS), respectively, fulfill similar protein functions as their bacterial counterparts. Their relative abundance, however, could reflect different mismatch specificity and/or specialisation for different tissues or organelles or developmental processes such as mitotic versus meiotic recombination.
To date, six different genes homologous to MutS have been isolated in yeast (yMSM), and their homologs have been found in mouse (mMSH) and human (hMSH), respectively. Encoded proteins yMSH2, yMSH3 and yMSH6 appear to be the main MutS homologs involved in MMR during mitosis and meiosis in yeast, where the complementary proteins MSH3 and MSH6 alternatively associate with MSH2 to recognise different mismatch substrates (Masischky et al., 1996, Genes Dev. 10, 407-420). Similar protein interactions have been demonstrated for the human homologs hMSH2, hMSH3 and hMSH6 (Acharya et al., 1996, PNAS 93, 13629-13634).
MutL homologs (MLH and PMS), recently reviewed by Modrich and Lahue (1996, Annual Rev. Biochem. 65, 101-133) have so far been found in yeast (yMLH1 and yPhVS1), mouse (mPMS2) and human (hMLH1, hPMS1 and hpMS2). The hPMS2 is a member of a family of at least 7 genes (Horii et al., 1994, Biochem. Biophys. Res. Commun. 204, 1257-1264) and its gene product is most closely related to yPMS1. Prolla et al. (1994, Science 265, 1091-1093) presented evidence for yPMS1 and yMLH1 to physically associate with each other and, together, to interact with the MutS homolog yMSH2 to form a ternary complex involved in mismatch substrate binding.
However, while medical interest in mismatch repair has prompted extensive research on MMR in bacteria, yeast and mammals, MMR genes have not been isolated from higher plants prior to the present invention and no attempts to adjust the plant MMR to plant breeding needs have been reported.
According to a first embodiment of the invention, there is provided an isolated and purified DNA molecule comprising a polynucleotide sequence encoding a polypeptide functionally involved in the DNA mismatch repair system of a plant. In one form of this embodiment, the invention provides an isolated and purified DNA molecule comprising a polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human. More particularly, the invention provides polynucleotide sequences encoding polypeptides which are homologous to the mismatch repair polypeptides MSH3 and MSH6 of Saccharomyces cerevisiae. Still, more particularly, the invention provides the coding sequences of the genes AtMSH3 and ArMSH6 of Arabidopsis thaliana, as defined hereinbelow, and polynucleotide sequences encoding polypeptides which are homologous to polypeptides encoded by AtMSH3 and ArMSH6.
According to a second embodiment of the invention, there is provided an isolated and purified polypeptide functionally involved in the DNA mismatch repair system of a plant, for example a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human such as a polypeptide encoded by the genes ArMSH3 or ArMSH6 of Arabidopsis thaliana, as defined hereinbelow.
According to a third embodiment of the invention, there is provided an isolated and purified DNA molecule comprising a polynucleotide sequence selected from the group consisting of (i) a sequence encoding a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence; and (ii) a sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant.
According to a fourth embodiment of the invention there is provided a chimeric gene comprising a DNA sequence selected from the group consisting of (i) a sequence encoding a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair polypeptide of a yeast or of a human and thereby disabling said plant polynucleotide sequence, and (ii) a sequence encoding a polypeptide capable of disrupting the DNA mismatch repair system of a plant; together with at least one regulation element capable of functioning in a plant cell. Examples of such regulation elements include constitutive, inducible, tissue type specific and cell type specific promoters such as 35S, NOS, PR1a, AoPR1 and DMC1. Typically, a chimeric gene of the fourth embodiment will also include at least one terminator sequence, more typically exactly one terminator sequence.
In the third and fourth embodiments, said interference, by said polynucleotide sequence, with the expression of a plant polynucleotide sequence encoding a polypeptide which is homologous to a mismatch repair peptide of a yeast or a human typically occurs by hybridisation or by co-suppression.
According to a fifth embodiment of the invention there is provided a plasmid or vector comprising a chimeric gene of the fourth embodiment. A vector of the fifth embodiment may be, for example, a viral vector or a bacterial vector.
According to a sixth embodiment of the invention, there is provided a plant cell stably transformed, transfected or electroporated with a plasmid or vector of the fifth embodiment.
According to seventh embodiment of the invention, there is provided a plant comprising a cell of the sixth embodiment.
According to an eighth embodiment of the invention, there is provided a process for at least partially inactivating a DNA mismatch repair system of a plant cell, comprising transforming or transfecting said plant cell with a DNA sequence of the third embodiment or a chimeric gene of the fourth embodiment or a plasmid or vector of the fifth embodiment, and causing said DNA sequence to express said polynucleotide or said polypeptide.
According to a ninth embodiment of the invention, there is provided a process for increasing genetic variation in a plant comprising obtaining a hybrid plant from a first plant and a second plant, or cells thereof, said first and second plants being genetically different; altering the mismatch repair system in said hybrid plant; permitting said hybrid plant to self-fertilise and produce offspring plants; and screening said offspring plants for plants in which homeologous recombination has occurred. For example, homeologous recombination may be evidenced by new genetic linkage of a desired characteristic trait or of a gene which contributes to a desired characteristic trait.
According to a tenth embodiment of the invention there is provided a process for obtaining a plant having a desired characteristic, comprising altering the mismatch repair system in a plant, cell or plurality of cells of a plant which does not have said desired characteristic, permitting mutations to persist in said cells to produce mutated plant cells, deriving plants from said mutated plant cells, and screening said plants for a plant having said desired characteristic.
In a preferred form of the ninth and tenth embodiments of the invention, the step of altering the mismatch repair system comprises introducing into said hybrid plant, plant, cell or cells a chimeric gene of the fourth embodiment and permitting the chimeric gene to express a polynucleotide which is capable of interfering with the expression of a plant polynucleotide sequence in a mismatch repair gene of the hybrid plant, plant, cell or cells, or a polypeptide capable of disrupting the DNA mismatch repair system of the hybrid plant or cells.
In other embodiments, the invention provides (a) an oligonucleotide capable of hybridising at 45xc2x0 C. under standard PCR conditions to a DNA molecule of the first embodiment; (b) an oligonucleotide capable of hybridising at 45xc2x0 C. under standard PCR conditions to the DNA of SEQ ID NO: 18 and (c) an oligonucleotide capable of hybridising at 45xc2x0 C. under standard PCR conditions to the DNA of SEQ ID NO:30; with the proviso that the oligonucleotide of (a), (b) and (c) is other than SEQ ID NO: 1 or SEQ ID NO:2.